Mayka Sánchez - Academia.edu (original) (raw)

Papers by Mayka Sánchez

he IRP/IRE regulatory system plays a crucial role in the post-transcriptional regulation of gene ... more he IRP/IRE regulatory system plays a crucial role in the post-transcriptional regulation of gene expression and its disruption results in human disease. Iron-responsive elements (IREs) are cis-acting regulatory motifs present in mRNAs that encode for proteins involved in iron metabolism. They function as binding sites for two related trans-acting factors, namely the iron regulatory proteins (IRP1 and IRP2). Among the cis-acting oligonucleotide patterns, the IRE is one of the best characterized. It is defined by a combination of RNA sequence and structure. However, currently available programs to predict IREs do not show a satisfactory level of sensitivity and fail to detect functional IREs. Here, we report an improved software for the prediction of IREs implemented as a user-friendly web-server tool. The SIREs web-server uses a simple data input interface and provides structure analysis, predicted RNA folds, folding energy data and an overall quality flag based on properties of well...

Haematologica

Iron refractory iron deficiency anemia is a hereditary recessive anemia due to a defect in the TM... more Iron refractory iron deficiency anemia is a hereditary recessive anemia due to a defect in the TMPRSS6 gene encoding Matriptase-2. This protein is a transmembrane serine protease that plays an essential role in down-regulating hepcidin, the key regulator of iron homeostasis. Hallmarks of this disease are microcytic hypochromic anemia, low transferrin saturation and normal/high serum hepcidin values. The anemia appears in the post-natal period, although in some cases it is only diagnosed in adulthood. The disease is refractory to oral iron treatment but shows a slow response to intravenous iron injections and partial correction of the anemia. To date, 40 different Matriptase-2 mutations have been reported, affecting all the functional domains of the large ectodomain of the protein. In vitro experiments on transfected cells suggest that Matriptase-2 cleaves Hemojuvelin, a major regulator of hepcidin expression and that this function is altered in this genetic form of anemia. In contra...

We have cloned and sequenced 1398bp of the rat HFE gene promoter region. The alignment of the rat... more We have cloned and sequenced 1398bp of the rat HFE gene promoter region. The alignment of the rat promoter HFE sequence with the HFE promoter sequence from human and mouse detected several highly conserved sequences present at orthologous or heterologous positions in the three species. Subsequent analysis of the conserved promoter sequences identified the presence of 10 novel transcription elements present in the promoter regions of the human, mouse and rat HFE genes (GATA, NF-IL6, AP1, AP2, CREB, PEA3, gamma-IRE, GFI1, HNF-3beta, HFH2). Different gel retardation analyses performed with rat-liver nuclear extracts have confirmed the presence of factors binding to some of these transcription elements. This represents the first data concerning the identification of potential transcriptional elements of the HFE promoter in these three species. The expression pattern of the transcription factors corresponding to the novel elements identified in the HFE promoter is consistent with the potential role of the HFE promoter in the transcription regulation and function of the HFE gene. Knowledge of the identified conserved elements in the HFE promoter from human, mouse and rat provides the basis for subsequent in-vitro or in-vivo studies leading to identification of the detailed mechanisms involved in the regulation of the iron metabolism and the design of potential future alternative therapies.

We have sequenced the 5' region of the SRY gene from human, chimpanzee, sheep, and mouse ... more We have sequenced the 5' region of the SRY gene from human, chimpanzee, sheep, and mouse and from four additional mammalian species, not previously characterized (gorilla, gazelle, rat, and guinea pig). In order to identify conserved DNA elements potentially involved in the regulation of the SRY gene, the newly determined sequences were analyzed and compared to all mammalian SRY promoter sequences available at present. Ten highly conserved potential regulatory elements have been identified in all 10 species (AP1, Barbie, GATA, Gfi1, cMyb, vMyb, NF1, Oct1, Sp1, and SRY). The known function of several of these regulatory elements fits well with the known expression of the SRY gene. However, except for the highly conserved coding HMG motif, only a short region close to the initiation of transcription in the human SRY is conserved in the exact position along the gene in all the species analyzed. This lack of sequence identity at the orthologous positions is consistent with the suggested rapid evolution of the SRY gene. This relative lack of homology contrasts with a high sequence identity of the putative regulatory sequences found within each taxonomic group of species (primates, bovids, and rodents), which supports a common mechanism of SRY expression and possibly also a similar function.

Endocrine, 2004

We initiated the present work to determine whether the presence of the HFE C282Y or H63D mutation... more We initiated the present work to determine whether the presence of the HFE C282Y or H63D mutations could be related to the clinical expression of diabetes mellitus type 2. Two hundred and twenty five type 2 consecutive diabetic patients were included and the HFE genotypes were determined. Younger ages of onset of diabetes as well as a longer duration of the disease were detected in patients carrying at least one C282Y allele (p = 0.007). An increased prevalence of retinopathy (p = 0.014) and of nephropathy (p = 0.04) were also detected in individuals carrying at least one C282Y allele in comparison with patients carrying the other alleles. The increased prevalence of retinopathy in C282Y carriers is related to the increased duration of the disease, but we not have detected that the prevalence of nephropathy is associated with the duration of the disease. However, multivariate logistic regression confirms that the prevalence of nephropathy is higher in the group of patients carrying at least one C282Y allele or the H63D/H63D genotype as compared to the group of patients with the wild-type (N/N) or the N/H63D genotype. To our knowledge our study is the first one to report an earlier age of onset in type 2 diabetic patients carrying HFE mutations.

Human mutation, 2014

Iron-refractory iron-deficiency anemia (IRIDA) is a rare autosomal-recessive disorder characteriz... more Iron-refractory iron-deficiency anemia (IRIDA) is a rare autosomal-recessive disorder characterized by hypochromic microcytic anemia, low transferrin saturation, and inappropriate high levels of the iron hormone hepcidin. The disease is caused by variants in the transmembrane protease serine 6 (TMPRSS6) gene that encodes the type II serine protease matriptase-2, a negative regulator of hepcidin transcription. Sequencing analysis of the TMPRSS6 gene in 21 new IRIDA patients from 16 families with different ethnic origin reveal 17 novel mutations, including the most frequent mutation in Southern Italy (p.W590R). Eight missense mutations were analyzed in vitro. All but the p.T287N variant impair matriptase-2 autoproteotylic activation, decrease the ability to cleave membrane HJV and inhibit the HJV-dependent hepcidin activation. Genotype-phenotype studies in IRIDA patients have been so far limited due to the relatively low number of described patients. Our genotype-phenotype correlation...

PLoS ONE, 2014

Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid seq... more Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzymeencoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 59 untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway.

Orphanet Journal of Rare Diseases, 2013

Background: Hereditary Hyperferritinaemia Cataract Syndrome (HHCS) is a rare autosomal dominant d... more Background: Hereditary Hyperferritinaemia Cataract Syndrome (HHCS) is a rare autosomal dominant disease characterized by increased serum ferritin levels and early onset of bilateral cataract. The disease is caused by mutations in the Iron-Responsive Element (IRE) located in the 5 0 untranslated region of L-Ferritin (FTL) mRNA, which post-transcriptionally regulates ferritin expression.

Nature Structural & Molecular Biology, 2007

Hypoxia stimulates erythropoiesis, the major iron-utilization pathway. We report the discovery of... more Hypoxia stimulates erythropoiesis, the major iron-utilization pathway. We report the discovery of a conserved, functional iron-responsive element (IRE) in the 5' untranslated region of the messenger RNA encoding endothelial PAS domain protein-1, EPAS1 (also called hypoxia-inducible factor-2alpha, HIF2alpha). Via this IRE, iron regulatory protein binding controls EPAS1 mRNA translation in response to cellular iron availability. Our results uncover a regulatory link that permits feedback control between iron availability and the expression of a key transcription factor promoting iron utilization. They also show that an IRE that is structurally distinct from, for example, the ferritin mRNA IRE and that has been missed by in silico approaches, can mediate mechanistically similar responses.

Nature Protocols, 2007

RNA-binding proteins (RBPs) frequently regulate the post-transcriptional fate of target mRNAs. To... more RNA-binding proteins (RBPs) frequently regulate the post-transcriptional fate of target mRNAs. To identify novel target mRNAs of RBPs, we incubate total RNA with recombinant RBP and immunoselect the messenger-ribonucleoproteins using a specific anti-RBP antibody. The mRNA composition of the supernatant and/or immunoprecipitated fraction is analyzed using dual-color microarrays in comparison with control reaction. From start to finish, the protocol takes approximately 6 d.

Journal of Molecular Medicine, 2012

Siderophores are best known as small iron binding molecules that facilitate microbial iron transp... more Siderophores are best known as small iron binding molecules that facilitate microbial iron transport. In our previous study we identified a siderophore-like molecule in mammalian cells and found that its biogenesis is evolutionarily conserved. A member of the short chain dehydrogenase family of reductases, 3-hydroxy butyrate dehydrogenase (BDH2) catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore. We have shown that depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of cellular iron and mitochondrial iron deficiency. These observations suggest that the mammalian siderophore is a critical regulator of cellular iron homeostasis and facilitates mitochondrial iron import. By utilizing bioinformatics, we identified an iron-responsive element (IRE; a stem-loop structure that regulates genes expression post-transcriptionally upon binding to iron regulatory proteins or IRPs) in the 3'-untranslated region of the human BDH2 (hBDH2) gene. In cultured cells as well as in patient samples we now demonstrate that the IRE confers iron-dependent regulation on hBDH2 and binds IRPs in RNA electrophoretic mobility shift assays. In addition, we show that the hBDH2 IRE associates with IRPs in cells and that abrogation of IRPs by RNAi eliminates the iron-dependent regulation of hBDH2 mRNA. The key physiologic implication is that iron-mediated post-transcriptional regulation of hBDH2 controls mitochondrial iron homeostasis in human cells. These observations provide a new and an unanticipated mechanism by which iron regulates its intracellular trafficking.

Journal of Hepatology, 2003

Background/Aims: Hereditary hemochromatosis is associated with homozygosity for C282Y mutation in... more Background/Aims: Hereditary hemochromatosis is associated with homozygosity for C282Y mutation in the HFE gene, elevated serum transferrin saturation and excess iron deposits throughout the body. We conducted a populationbased study in Spain to asses the prevalence of the HFE mutations and their effect on iron parameters.

Journal of Biological Chemistry, 2006

Iron regulatory proteins (IRPs) 1 and 2 post-transcriptionally control mammalian iron homeostasis... more Iron regulatory proteins (IRPs) 1 and 2 post-transcriptionally control mammalian iron homeostasis by binding to iron-responsive elements (IREs), conserved RNA stem-loop structures located in the 5-or 3-untranslated regions of genes involved in iron metabolism (e.g. FTH1, FTL, and TFRC). To identify novel IRE-containing mRNAs, we integrated biochemical, biocomputational, and microarray-based experimental approaches. IRP/ IRE messenger ribonucleoproteins were immunoselected, and their mRNA composition was analyzed using an IronChip microarray enriched for genes predicted computationally to contain IRE-like motifs. Among different candidates, this report focuses on a novel IRE located in the 3-untranslated region of the cell division cycle 14A mRNA. We show that this IRE motif efficiently binds both IRP1 and IRP2. Differential splicing of cell division cycle 14A produces IRE-and non-IRE-containing mRNA isoforms. Interestingly, only the expression of the IREcontaining mRNA isoforms is selectively increased by cellular iron deficiency. This work describes a new experimental strategy to explore the IRE/IRP regulatory network and uncovers a previously unrecognized regulatory link between iron metabolism and the cell cycle.

Gene, 1998

We have cloned and sequenced 1398bp of the rat HFE gene promoter region. The alignment of the rat... more We have cloned and sequenced 1398bp of the rat HFE gene promoter region. The alignment of the rat promoter HFE sequence with the HFE promoter sequence from human and mouse detected several highly conserved sequences present at orthologous or heterologous positions in the three species. Subsequent analysis of the conserved promoter sequences identified the presence of 10 novel transcription elements present in the promoter regions of the human, mouse and rat HFE genes (GATA, NF-IL6, AP1, AP2, CREB, PEA3, gamma-IRE, GFI1, HNF-3beta, HFH2). Different gel retardation analyses performed with rat-liver nuclear extracts have confirmed the presence of factors binding to some of these transcription elements. This represents the first data concerning the identification of potential transcriptional elements of the HFE promoter in these three species. The expression pattern of the transcription factors corresponding to the novel elements identified in the HFE promoter is consistent with the potential role of the HFE promoter in the transcription regulation and function of the HFE gene. Knowledge of the identified conserved elements in the HFE promoter from human, mouse and rat provides the basis for subsequent in-vitro or in-vivo studies leading to identification of the detailed mechanisms involved in the regulation of the iron metabolism and the design of potential future alternative therapies.

European Journal of Gastroenterology & Hepatology, 1999

A mutation (Cys282Tyr) of the HFE gene has recently been reported to be present in most of the pa... more A mutation (Cys282Tyr) of the HFE gene has recently been reported to be present in most of the patients with hereditary hemochromatosis of Northern European ancestry, but in a lower frequency in Italy. No data are so far available on the prevalence of these mutations in Spain. Therefore, we initiated the present study to determine if the reported Cys282Tyr HFE mutation is also the main cause of hereditary hemochromatosis in Spain. In addition, we investigated the presence of the His63Asp HFE mutation in patients and in controls. Thirty-one hereditary hemochromatosis patients and 485 controls were screened for the Cys282Tyr and the His63Asp mutations, using polymerase chain reaction amplification of genomic DNA, followed by digestion with the restriction enzymes Rsa I or Dpn II, respectively, and the separation of the products by electrophoresis. Twenty-seven out of 31 (87.1%) hereditary hemochromatosis patients were homozygous for the Cys282Tyr mutation. None of the patients was homozygous for the His63Asp mutation, and two patients (6.5%) were compound heterozygous (Cys282Tyr/His63Asp). Only one of 512 (0.2%) controls was homozygous for the Cys282Tyr mutation, and 29 (5.7%) were heterozygous. The Cys282Tyr mutation is present with an allelic frequency of 90.3+/-7.5% in patients with hereditary hemochromatosis and 3.0+/-1.1% in controls. Twenty out of 487 (4.1%) controls were His63Asp homozygous, while 171 (35.1%) were heterozygous. The His63Asp mutation is present with an allelic frequency of 21.7+/-2.7% in controls. The high frequency of the Cys282Tyr mutation in hereditary hemochromatosis patients indicates that this mutation is the most common defect associated with hereditary hemochromatosis in Spain. The finding of some patients with the wild genotype at position 282 suggests the existence of other changes in the HFE gene or in other loci involved in the disease. We have found one of the highest allelic frequencies reported for the His63Asp mutation in our controls (21.7+/-2.7%).

BMC Bioinformatics, 2010

Background: Gene expression studies greatly contribute to our understanding of complex relationsh... more Background: Gene expression studies greatly contribute to our understanding of complex relationships in gene regulatory networks. However, the complexity of array design, production and manipulations are limiting factors, affecting data quality. The use of customized DNA microarrays improves overall data quality in many situations, however, only if for these specifically designed microarrays analysis tools are available. Results: The IronChip Evaluation Package (ICEP) is a collection of Perl utilities and an easy to use data evaluation pipeline for the analysis of microarray data with a focus on data quality of custom-designed microarrays. The package has been developed for the statistical and bioinformatical analysis of the custom cDNA microarray IronChip but can be easily adapted for other cDNA or oligonucleotide-based designed microarray platforms. ICEP uses decision tree-based algorithms to assign quality flags and performs robust analysis based on chip design properties regarding multiple repetitions, ratio cut-off, background and negative controls. Conclusions: ICEP is a stand-alone Windows application to obtain optimal data quality from custom-designed microarrays and is freely available here (see "Additional Files" section) and at: http://www.alice-dsl.net/evgeniy. vainshtein/ICEP/

Blood, 2011

Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metab... more Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition was determined using microarrays. We identify 35 novel mRNAs that bind both IRP1 and IRP2, and we also report for the first time cellular mRNAs with exclusive specificity for IRP1 or IRP2. To further explore cellular iron metabolism at a system-wide level, we undertook proteomic analysis by pulsed stable isotope labeling by amino acids in cell culture in an iron-modulated mouse hepatic cell line and in bone marrow-derived macrophages from IRP1- and IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with iron-dependent regulation, IRP-dependent regulation, or both.

Molecular Genetics & Genomic Medicine, 2015

he IRP/IRE regulatory system plays a crucial role in the post-transcriptional regulation of gene ... more he IRP/IRE regulatory system plays a crucial role in the post-transcriptional regulation of gene expression and its disruption results in human disease. Iron-responsive elements (IREs) are cis-acting regulatory motifs present in mRNAs that encode for proteins involved in iron metabolism. They function as binding sites for two related trans-acting factors, namely the iron regulatory proteins (IRP1 and IRP2). Among the cis-acting oligonucleotide patterns, the IRE is one of the best characterized. It is defined by a combination of RNA sequence and structure. However, currently available programs to predict IREs do not show a satisfactory level of sensitivity and fail to detect functional IREs. Here, we report an improved software for the prediction of IREs implemented as a user-friendly web-server tool. The SIREs web-server uses a simple data input interface and provides structure analysis, predicted RNA folds, folding energy data and an overall quality flag based on properties of well...

Haematologica

Iron refractory iron deficiency anemia is a hereditary recessive anemia due to a defect in the TM... more Iron refractory iron deficiency anemia is a hereditary recessive anemia due to a defect in the TMPRSS6 gene encoding Matriptase-2. This protein is a transmembrane serine protease that plays an essential role in down-regulating hepcidin, the key regulator of iron homeostasis. Hallmarks of this disease are microcytic hypochromic anemia, low transferrin saturation and normal/high serum hepcidin values. The anemia appears in the post-natal period, although in some cases it is only diagnosed in adulthood. The disease is refractory to oral iron treatment but shows a slow response to intravenous iron injections and partial correction of the anemia. To date, 40 different Matriptase-2 mutations have been reported, affecting all the functional domains of the large ectodomain of the protein. In vitro experiments on transfected cells suggest that Matriptase-2 cleaves Hemojuvelin, a major regulator of hepcidin expression and that this function is altered in this genetic form of anemia. In contra...

We have cloned and sequenced 1398bp of the rat HFE gene promoter region. The alignment of the rat... more We have cloned and sequenced 1398bp of the rat HFE gene promoter region. The alignment of the rat promoter HFE sequence with the HFE promoter sequence from human and mouse detected several highly conserved sequences present at orthologous or heterologous positions in the three species. Subsequent analysis of the conserved promoter sequences identified the presence of 10 novel transcription elements present in the promoter regions of the human, mouse and rat HFE genes (GATA, NF-IL6, AP1, AP2, CREB, PEA3, gamma-IRE, GFI1, HNF-3beta, HFH2). Different gel retardation analyses performed with rat-liver nuclear extracts have confirmed the presence of factors binding to some of these transcription elements. This represents the first data concerning the identification of potential transcriptional elements of the HFE promoter in these three species. The expression pattern of the transcription factors corresponding to the novel elements identified in the HFE promoter is consistent with the potential role of the HFE promoter in the transcription regulation and function of the HFE gene. Knowledge of the identified conserved elements in the HFE promoter from human, mouse and rat provides the basis for subsequent in-vitro or in-vivo studies leading to identification of the detailed mechanisms involved in the regulation of the iron metabolism and the design of potential future alternative therapies.

We have sequenced the 5' region of the SRY gene from human, chimpanzee, sheep, and mouse ... more We have sequenced the 5' region of the SRY gene from human, chimpanzee, sheep, and mouse and from four additional mammalian species, not previously characterized (gorilla, gazelle, rat, and guinea pig). In order to identify conserved DNA elements potentially involved in the regulation of the SRY gene, the newly determined sequences were analyzed and compared to all mammalian SRY promoter sequences available at present. Ten highly conserved potential regulatory elements have been identified in all 10 species (AP1, Barbie, GATA, Gfi1, cMyb, vMyb, NF1, Oct1, Sp1, and SRY). The known function of several of these regulatory elements fits well with the known expression of the SRY gene. However, except for the highly conserved coding HMG motif, only a short region close to the initiation of transcription in the human SRY is conserved in the exact position along the gene in all the species analyzed. This lack of sequence identity at the orthologous positions is consistent with the suggested rapid evolution of the SRY gene. This relative lack of homology contrasts with a high sequence identity of the putative regulatory sequences found within each taxonomic group of species (primates, bovids, and rodents), which supports a common mechanism of SRY expression and possibly also a similar function.

Endocrine, 2004

We initiated the present work to determine whether the presence of the HFE C282Y or H63D mutation... more We initiated the present work to determine whether the presence of the HFE C282Y or H63D mutations could be related to the clinical expression of diabetes mellitus type 2. Two hundred and twenty five type 2 consecutive diabetic patients were included and the HFE genotypes were determined. Younger ages of onset of diabetes as well as a longer duration of the disease were detected in patients carrying at least one C282Y allele (p = 0.007). An increased prevalence of retinopathy (p = 0.014) and of nephropathy (p = 0.04) were also detected in individuals carrying at least one C282Y allele in comparison with patients carrying the other alleles. The increased prevalence of retinopathy in C282Y carriers is related to the increased duration of the disease, but we not have detected that the prevalence of nephropathy is associated with the duration of the disease. However, multivariate logistic regression confirms that the prevalence of nephropathy is higher in the group of patients carrying at least one C282Y allele or the H63D/H63D genotype as compared to the group of patients with the wild-type (N/N) or the N/H63D genotype. To our knowledge our study is the first one to report an earlier age of onset in type 2 diabetic patients carrying HFE mutations.

Human mutation, 2014

Iron-refractory iron-deficiency anemia (IRIDA) is a rare autosomal-recessive disorder characteriz... more Iron-refractory iron-deficiency anemia (IRIDA) is a rare autosomal-recessive disorder characterized by hypochromic microcytic anemia, low transferrin saturation, and inappropriate high levels of the iron hormone hepcidin. The disease is caused by variants in the transmembrane protease serine 6 (TMPRSS6) gene that encodes the type II serine protease matriptase-2, a negative regulator of hepcidin transcription. Sequencing analysis of the TMPRSS6 gene in 21 new IRIDA patients from 16 families with different ethnic origin reveal 17 novel mutations, including the most frequent mutation in Southern Italy (p.W590R). Eight missense mutations were analyzed in vitro. All but the p.T287N variant impair matriptase-2 autoproteotylic activation, decrease the ability to cleave membrane HJV and inhibit the HJV-dependent hepcidin activation. Genotype-phenotype studies in IRIDA patients have been so far limited due to the relatively low number of described patients. Our genotype-phenotype correlation...

PLoS ONE, 2014

Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid seq... more Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzymeencoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 59 untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway.

Orphanet Journal of Rare Diseases, 2013

Background: Hereditary Hyperferritinaemia Cataract Syndrome (HHCS) is a rare autosomal dominant d... more Background: Hereditary Hyperferritinaemia Cataract Syndrome (HHCS) is a rare autosomal dominant disease characterized by increased serum ferritin levels and early onset of bilateral cataract. The disease is caused by mutations in the Iron-Responsive Element (IRE) located in the 5 0 untranslated region of L-Ferritin (FTL) mRNA, which post-transcriptionally regulates ferritin expression.

Nature Structural & Molecular Biology, 2007

Hypoxia stimulates erythropoiesis, the major iron-utilization pathway. We report the discovery of... more Hypoxia stimulates erythropoiesis, the major iron-utilization pathway. We report the discovery of a conserved, functional iron-responsive element (IRE) in the 5' untranslated region of the messenger RNA encoding endothelial PAS domain protein-1, EPAS1 (also called hypoxia-inducible factor-2alpha, HIF2alpha). Via this IRE, iron regulatory protein binding controls EPAS1 mRNA translation in response to cellular iron availability. Our results uncover a regulatory link that permits feedback control between iron availability and the expression of a key transcription factor promoting iron utilization. They also show that an IRE that is structurally distinct from, for example, the ferritin mRNA IRE and that has been missed by in silico approaches, can mediate mechanistically similar responses.

Nature Protocols, 2007

RNA-binding proteins (RBPs) frequently regulate the post-transcriptional fate of target mRNAs. To... more RNA-binding proteins (RBPs) frequently regulate the post-transcriptional fate of target mRNAs. To identify novel target mRNAs of RBPs, we incubate total RNA with recombinant RBP and immunoselect the messenger-ribonucleoproteins using a specific anti-RBP antibody. The mRNA composition of the supernatant and/or immunoprecipitated fraction is analyzed using dual-color microarrays in comparison with control reaction. From start to finish, the protocol takes approximately 6 d.

Journal of Molecular Medicine, 2012

Siderophores are best known as small iron binding molecules that facilitate microbial iron transp... more Siderophores are best known as small iron binding molecules that facilitate microbial iron transport. In our previous study we identified a siderophore-like molecule in mammalian cells and found that its biogenesis is evolutionarily conserved. A member of the short chain dehydrogenase family of reductases, 3-hydroxy butyrate dehydrogenase (BDH2) catalyzes a rate-limiting step in the biogenesis of the mammalian siderophore. We have shown that depletion of the mammalian siderophore by inhibiting expression of bdh2 results in abnormal accumulation of cellular iron and mitochondrial iron deficiency. These observations suggest that the mammalian siderophore is a critical regulator of cellular iron homeostasis and facilitates mitochondrial iron import. By utilizing bioinformatics, we identified an iron-responsive element (IRE; a stem-loop structure that regulates genes expression post-transcriptionally upon binding to iron regulatory proteins or IRPs) in the 3'-untranslated region of the human BDH2 (hBDH2) gene. In cultured cells as well as in patient samples we now demonstrate that the IRE confers iron-dependent regulation on hBDH2 and binds IRPs in RNA electrophoretic mobility shift assays. In addition, we show that the hBDH2 IRE associates with IRPs in cells and that abrogation of IRPs by RNAi eliminates the iron-dependent regulation of hBDH2 mRNA. The key physiologic implication is that iron-mediated post-transcriptional regulation of hBDH2 controls mitochondrial iron homeostasis in human cells. These observations provide a new and an unanticipated mechanism by which iron regulates its intracellular trafficking.

Journal of Hepatology, 2003

Background/Aims: Hereditary hemochromatosis is associated with homozygosity for C282Y mutation in... more Background/Aims: Hereditary hemochromatosis is associated with homozygosity for C282Y mutation in the HFE gene, elevated serum transferrin saturation and excess iron deposits throughout the body. We conducted a populationbased study in Spain to asses the prevalence of the HFE mutations and their effect on iron parameters.

Journal of Biological Chemistry, 2006

Iron regulatory proteins (IRPs) 1 and 2 post-transcriptionally control mammalian iron homeostasis... more Iron regulatory proteins (IRPs) 1 and 2 post-transcriptionally control mammalian iron homeostasis by binding to iron-responsive elements (IREs), conserved RNA stem-loop structures located in the 5-or 3-untranslated regions of genes involved in iron metabolism (e.g. FTH1, FTL, and TFRC). To identify novel IRE-containing mRNAs, we integrated biochemical, biocomputational, and microarray-based experimental approaches. IRP/ IRE messenger ribonucleoproteins were immunoselected, and their mRNA composition was analyzed using an IronChip microarray enriched for genes predicted computationally to contain IRE-like motifs. Among different candidates, this report focuses on a novel IRE located in the 3-untranslated region of the cell division cycle 14A mRNA. We show that this IRE motif efficiently binds both IRP1 and IRP2. Differential splicing of cell division cycle 14A produces IRE-and non-IRE-containing mRNA isoforms. Interestingly, only the expression of the IREcontaining mRNA isoforms is selectively increased by cellular iron deficiency. This work describes a new experimental strategy to explore the IRE/IRP regulatory network and uncovers a previously unrecognized regulatory link between iron metabolism and the cell cycle.

Gene, 1998

We have cloned and sequenced 1398bp of the rat HFE gene promoter region. The alignment of the rat... more We have cloned and sequenced 1398bp of the rat HFE gene promoter region. The alignment of the rat promoter HFE sequence with the HFE promoter sequence from human and mouse detected several highly conserved sequences present at orthologous or heterologous positions in the three species. Subsequent analysis of the conserved promoter sequences identified the presence of 10 novel transcription elements present in the promoter regions of the human, mouse and rat HFE genes (GATA, NF-IL6, AP1, AP2, CREB, PEA3, gamma-IRE, GFI1, HNF-3beta, HFH2). Different gel retardation analyses performed with rat-liver nuclear extracts have confirmed the presence of factors binding to some of these transcription elements. This represents the first data concerning the identification of potential transcriptional elements of the HFE promoter in these three species. The expression pattern of the transcription factors corresponding to the novel elements identified in the HFE promoter is consistent with the potential role of the HFE promoter in the transcription regulation and function of the HFE gene. Knowledge of the identified conserved elements in the HFE promoter from human, mouse and rat provides the basis for subsequent in-vitro or in-vivo studies leading to identification of the detailed mechanisms involved in the regulation of the iron metabolism and the design of potential future alternative therapies.

European Journal of Gastroenterology & Hepatology, 1999

A mutation (Cys282Tyr) of the HFE gene has recently been reported to be present in most of the pa... more A mutation (Cys282Tyr) of the HFE gene has recently been reported to be present in most of the patients with hereditary hemochromatosis of Northern European ancestry, but in a lower frequency in Italy. No data are so far available on the prevalence of these mutations in Spain. Therefore, we initiated the present study to determine if the reported Cys282Tyr HFE mutation is also the main cause of hereditary hemochromatosis in Spain. In addition, we investigated the presence of the His63Asp HFE mutation in patients and in controls. Thirty-one hereditary hemochromatosis patients and 485 controls were screened for the Cys282Tyr and the His63Asp mutations, using polymerase chain reaction amplification of genomic DNA, followed by digestion with the restriction enzymes Rsa I or Dpn II, respectively, and the separation of the products by electrophoresis. Twenty-seven out of 31 (87.1%) hereditary hemochromatosis patients were homozygous for the Cys282Tyr mutation. None of the patients was homozygous for the His63Asp mutation, and two patients (6.5%) were compound heterozygous (Cys282Tyr/His63Asp). Only one of 512 (0.2%) controls was homozygous for the Cys282Tyr mutation, and 29 (5.7%) were heterozygous. The Cys282Tyr mutation is present with an allelic frequency of 90.3+/-7.5% in patients with hereditary hemochromatosis and 3.0+/-1.1% in controls. Twenty out of 487 (4.1%) controls were His63Asp homozygous, while 171 (35.1%) were heterozygous. The His63Asp mutation is present with an allelic frequency of 21.7+/-2.7% in controls. The high frequency of the Cys282Tyr mutation in hereditary hemochromatosis patients indicates that this mutation is the most common defect associated with hereditary hemochromatosis in Spain. The finding of some patients with the wild genotype at position 282 suggests the existence of other changes in the HFE gene or in other loci involved in the disease. We have found one of the highest allelic frequencies reported for the His63Asp mutation in our controls (21.7+/-2.7%).

BMC Bioinformatics, 2010

Background: Gene expression studies greatly contribute to our understanding of complex relationsh... more Background: Gene expression studies greatly contribute to our understanding of complex relationships in gene regulatory networks. However, the complexity of array design, production and manipulations are limiting factors, affecting data quality. The use of customized DNA microarrays improves overall data quality in many situations, however, only if for these specifically designed microarrays analysis tools are available. Results: The IronChip Evaluation Package (ICEP) is a collection of Perl utilities and an easy to use data evaluation pipeline for the analysis of microarray data with a focus on data quality of custom-designed microarrays. The package has been developed for the statistical and bioinformatical analysis of the custom cDNA microarray IronChip but can be easily adapted for other cDNA or oligonucleotide-based designed microarray platforms. ICEP uses decision tree-based algorithms to assign quality flags and performs robust analysis based on chip design properties regarding multiple repetitions, ratio cut-off, background and negative controls. Conclusions: ICEP is a stand-alone Windows application to obtain optimal data quality from custom-designed microarrays and is freely available here (see "Additional Files" section) and at: http://www.alice-dsl.net/evgeniy. vainshtein/ICEP/

Blood, 2011

Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metab... more Iron regulatory proteins (IRPs) 1 and 2 are RNA-binding proteins that control cellular iron metabolism by binding to conserved RNA motifs called iron-responsive elements (IREs). The currently known IRP-binding mRNAs encode proteins involved in iron uptake, storage, and release as well as heme synthesis. To systematically define the IRE/IRP regulatory network on a transcriptome-wide scale, IRP1/IRE and IRP2/IRE messenger ribonucleoprotein complexes were immunoselected, and the mRNA composition was determined using microarrays. We identify 35 novel mRNAs that bind both IRP1 and IRP2, and we also report for the first time cellular mRNAs with exclusive specificity for IRP1 or IRP2. To further explore cellular iron metabolism at a system-wide level, we undertook proteomic analysis by pulsed stable isotope labeling by amino acids in cell culture in an iron-modulated mouse hepatic cell line and in bone marrow-derived macrophages from IRP1- and IRP2-deficient mice. This work investigates cellular iron metabolism in unprecedented depth and defines a wide network of mRNAs and proteins with iron-dependent regulation, IRP-dependent regulation, or both.

Molecular Genetics & Genomic Medicine, 2015